The broad objectives of this research program are the acquisition of a better understanding of the detailed enzymatic mechanisms involved in the transformation of steroid hormones in microbial and mammalian systems. The principal reactions to be studied are: 1. The delta5-3-ketosteroid isomerase reaction which plays a critical role in the biosynthesis of steroid hormones, and 2. Steroid oxidations by nicotinamide adenine dinucleotide-linked hydroxy-steroid dehydrogenases. Specific attention will be accorded to: 1. The basic mechanisms of the steroid transformations including the purification of the enzymes involved, their molecular properties, cofactor requirements and catalytic mechanisms. 2. Development of procedures of affinity chromatography for the purification of these enzymes and for the analysis of their catalytic processes. 3. Description of substrate and inhibitor specificities of steroid transforming enzymes. 4. Examination of the properties of isofunctional enzymes in different species, tissues and intracellular locations. 5. Studies of specific and tissue-selective inhibitors of these enzymatic reactions with a view to establishing their functional significance and identifying rate-limiting steps in reaction sequences. Particular effort will be devoted to the study of active-site-directed irreversible enzyme inhibitors capable of covalent interaction with the active sites of these enzymes. Enzyme-generated reactive derivatives offer particularly favorable features for achieving selectivity of inhibition. 6. The complete description of the details of the active site of the crystalline delta5-3-ketosteroid isomerase by affinity labeling and by X-ray crystallography. 7. The evaluation of these inhibitors in purified steroid-metabolizing enzyme preparations as well as in more highly organized systems with a view to perturbing normal or abnormal endocrine function.